Use of 2,5-Dihydroxybenzenesulfonic Acid in the Manufacturing of Medicines, Applicable to the Treatment of Angiodependent Diseases

ABSTRACT

Provided is the use of 2,5-dihydroxybenzenesulfonic acid in the production of medicaments for the treatment of angiodependent diseases. More specifically, described is the use of the aforesaid compound and, in particular, the calcium and potassium salts thereof, for the treatment of two angiodependent diseases, which present a reduction in the apoptosis, namely cancer and psoriasis. Also described is the antiproliferative, antimigratory, antiangiogenic and proapoptotic capacity of said family of compounds in non-quiescent cells. In addition, described is the potentiating effect of said compounds on known cytostatic medicines in the treatment of tumours and, specifically, on gliomas. Additionally, the therapeutic efficacy of said compounds, based on the combined anti proliferative, antiangiogenic and proapaptotic capacities thereof, in the treatment of chronic psoriatic plaques is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/169,781, filedJun. 27, 2011, which is a continuation of U.S. Ser. No. 10/588,166 filedAug. 7, 2008, and issued as U.S. Pat. No. 7,968,531 on Jun. 28, 2011,which is a national stage filing under 35 U.S.C. §371 of InternationalApplication Number PCT/ES2005/70017, filed Feb. 16, 2005, which claimsthe benefit of priority under 35 U.S.C. §119 of ES Application NumberP200400371, filed Feb. 17, 2004. The foregoing applications, and alldocuments cited therein, are hereby incorporated herein by reference intheir entirety.

TECHNICAL FIELD

The invention relates to a pharmaceutical composition that includes the2,5-dihydroxybenzenesulfonic acid, and its employment in the preparationof a medicine for treatment of diseases characterized by an intense cellproliferation, vascularization (angiodependent diseases) and morespecifically angiodependent diseases also having reduction of theapoptosis, as it is the case for example in cancer or psoriasis.

BACKGROUND

Malignant tumors are characterized, besides from the uncontrolledcellular proliferation, by their capacity to invade normal peritumoraltissues. Tumor invasion is a complex process developed according to thefollowing consecutive stages: a) adhesion of the tumor cells to proteinsof the extra-cellular matrix; b) degradation of the proteins of theextra-cellular matrix by proteases that create extra-cellular spacesthat the tumor cells use to, c) migrate through a dynamic and complexmechanism that requires synthesis of new portions of the cytoplasmicmembrane and reorganization of the cytoskeleton (Giese A, Westphal M.Neurosurgery 1996; 39: 235-252). The cells that from the tumor massinvade the normal peritumoral-tissue have their genetic program ofcellular death disabled and therefore, the tumor cells that migrate toinvade the peritumoral intact tissues, elude the apoptosis (Mariani I etal. Clin Cancer Res 7:2480-2489, 2001). When the grouped tumor cellsreach 2 to 3 mm³ volume, the tumor cells synthesize large amounts ofangiogenic factors to counteract the hypoxic situation of this primarytumor, (Folkman J. N. Engl J Med 285: 1182-1186, 1971; Carmeliet P, JainR K. Nature 407: 249-257, 2000; Yancopoulos G D et al. Nature 407:242-248, 2000) that activate the peritumoral blood vessels so that theyform new blood vessels (angiogenesis) that invade the tumor to supplythe oxygen and the nutrients and eliminate products from the tumorcatabolism. The same cellular processes that occur during the tumorinvasion (motility and absence of apoptosis) occur centripetally duringtumor angiogenesis. Therefore, the inhibition of the invasive capacityof the tumor cells and of the endothelial cells should produce a delayin tumor growth by inhibiting the tumor expansion, reducing angiogenesisand promoting apoptosis. Therefore, an effective treatment againstcancer should inhibit the migration, the angiogenesis and increaseapoptosis without producing these effects in normal cells.

There are numerous anti-tumor and antiangiogenic agents at variousstages of clinical development in oncology (Brem S. Cancer Control 6:436-458, 1999), of which a significant number are peptides that the bodyuses to counteract the effect of the positive regulators of angiogenesis(Hagerdom M, Bikfalvi A. Crit Rev One Hemat 34: 89-110, 2000). However,when these peptides are compared with compounds with a significantlylower molecular weight, their pharmacological inconveniences becomeevident. On the other hand, it has been proven that different syntheticcompounds containing aromatic rings in their molecular structure andacting as inhibitors of the mitogenic activity induced by growth factorsare cytotoxic for quiescent or non tumor cells (Lozano R M J Mol Biol 28I: 899-9115, 1998). Therefore, there is still need to find compoundswith anti-tumor, antiangiogenic and proapoptotic activity with lowtoxicity for intact, quiescent, non tumor cells. There is presently agreat interest for the search of new therapeutic indications for oldmedicines. In this connection, it has been recently proven thatdifferent antibiotics, besides from their antimicrobial activity, haveantiproliferative effects, such in the case of rapamycin (Morice M C etal. N Engl J Med 346: 1773-1780, 2002), or of the neomycin (Cuevas P. etal. Neural Res 224: 389-391, 2002); or are useful as anxiolytics such asnorfloxacin (fluoroquinolone) (Johnstone T B et al. Nat Med 10; 31-32,2004).

Psoriasis is an angiodependent chronic disease that affects 2-3% of theworld population and is characterized by epidermic hyperplasia,dermo-epidermic infiltration of inflammatory cells and T lymphocytes,and a very evident development of vascularization, together with areduction of the cell death due to apoptosis (Kocak M et al, Int JDermatol 42: 789-793, 2003). Presently, there is no curative treatmentfor psoriasis. The antipsoriatic treatment may be topical or systemic,depending on the extension and severity of the disease. The mostly usedanti psoriatic topical therapy consists of different types ofcorticoids, but the extended use of these compounds is associated withskin atrophy, stretch marks and telangiectasia (Baker B S, Fry L. Cutis1999; 64: 315-318). The systemic therapy with immunosuppressantmedicines is associated to very severe side effects (Wolina V. et al.Clin Rheumatol 2001: 20: 406-410). For example, the use of cyclosporinefor treatment of psoriasis may produce nephrotoxicity (interstitialfibrosis and tubular atrophy), hypertension, hypomagnesaemia,hypercalcemia and hepatic dysfunction (Travis L, Weinberg J M. Drugs ofToday 2002; 38: 847-865). The standing use of another immunosuppressantmedicine for treatment of psoriasis, tacrolirnus, may producehypertension, nephrotoxicity and immunosuppression (Jegasothy B V et al.Arch Dermatol 1992; 128: 781-785). It has been recently described thatthe topic application of the tacrolimus immunosuppressant acceleratescarcinogenesis in mouse skin (Niwa Y, Terashima T, Sumi H. B J Dermatol2003; 149: 960-967). Therefore, there is need for new antipsoriaticcompounds proving to be efficient without producing evident side effectssuch as those associated with the most common anti-psoriatic compounds.

The 2,5-dihydroxybenzenesulfonic acid is a derivative of the2,5-dihydroxybenzoic acid, pharmacologically prescribed in the form ofdifferent salts (mainly calcium, potassium, and magnesium), whichprovides stability. The 2,5-dihydroxybenzenesulfonic acid has been usedsince the 70's as an oral vasculotropic medicine.

The 2,5-dihydroxybenzenesulfonic acid inhibits platelet aggregation,increase of capilar permeability and blood viscosity in patients withdiabetic retinopathy (Bayer J. et al. Dtsch. Mod Wschr 1980; 46:160-1608; Banarroch L S. et al. Ophthalmic Res 1985; 17; 131-138; MichalM, Giessinger N. Thromb Res 1988; 51: 593-605). The metabolism and thepharmacokinetics of this compound in the human being is known since year1974 (Benakis A. et al. Therapie 1974; 29: 211-219). Recent experimentshave proven that the 2,5-dihydroxybenzenesulfonic acid increases theactivity of the endothelial isoform of the nitric oxide synthase[endothelial nitric oxyde synthase (eNOS)] in rat endothelial cellswithout producing cytotoxic effects (Suscheck C. et al. Bt J Pharmacal1997; 122: 1502-1508). In addition, the 2,5-dihydroxybenzenesulfonicacid potentiates the in vitro relaxation of human penile arteries(Angulo J et al. Br J Pharmacol 2003; 139: 854-862). There isexperimental evidence that the 2,5-dihydroxybenzenesulfonic acid(formulated as a calcium or magnesium salts) possesses in vitroantioxidant activities (Brunet J et al. Fundam Clin Pharmacol 12:205-212, 1998).

SUMMARY

The present invention is based on the discovery of new activities of the2,5-dihydroxybenzenesulfonic acid and/or its salts, associated to theirantiproliferative, anti migratory, antiangiogenic and proapoptoticcapacity in non quiescent cells, activities that combined, justify theiremployment as a useful compound for treatment of angiodependent diseasessuch as the case of cancer, characterized by hyperproliferation, cellinvasion and excessive angiogenesis, together with a deficit in celldeath due to apoptosis, without causing toxicity for non-tumor intact orquiescent cells. Gliomic tumor cells have been used in experimentsbecause gliomas are very invasive tumors with a significant angiogeniccapacity and a significant apoptotic deficit (Merzak A, Pilkington G J.Cancer Metastasis Rev 16: 155-177, 1997).

The present invention is also based on the proven fact that the2,5-dihydroxybenzenesulfonic acid and/or its salts possess, in acombined form, antipoliferative, antiangiogenic, and proapoptoticeffects and therefore its therapeutic efficacy has been evaluated inchronic psoriatic plaques characterized by epidermic hyperproliferation,acute dermal angiogenesis and apoptotic deficit (Karasek M A, Cutis 64:319-322, 1999).

This invention relates then to the search of new treatments for cancerand other angiodependent diseases and it is based on the fact that the2,5-dihydroxybenzene sulfonic acid and/or its salts have proven theircapacity to inhibit growth and migration and induce the apoptosis in invitro tumor cells as well as the capacity to inhibit the in vivoangiogenesis induced by fibroblast growth factor (FGF). Therefore, dueto the combination of these abilities, the mentioned compounds becomeuseful for the treatment of malignant tumors and hematologicalneoplastic diseases as well as for treatment of other severevascularization related pathologies (angiodependent diseases).

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are graphs depicting treatment with differentconcentrations of a compound according to one embodiment producing adose-dependent inhibition of cell proliferation. FIG. 1A depicts 88%inhibition with a concentration of 100 μM of the calcium salt of the2,5-dihydroxybenzenesulfonic acid. FIG. 1B depicts 74% inhibition withthe same concentration of the potassium salt of the2,5-dihydroxybenzenesulfonic acid;

FIG. 2A shows the image of a C6 cell culture after 48 hours withouttreatment; FIG. 2B shows the image of a C6 cell culture after 48 hoursof treatment with a concentration of 50 μM of the calcium salt of the2,5-dihydroxybenzenesulfonic acid; FIG. 2C shows the image of a C6 cellculture after 48 hours of treatment with 100 μM of the potassium salt ofthe acid;

FIGS. 3A and 3B are representative graphs depicting theantiproliferative effect of the 2,5-dihydroxybenzenesulfonic acid. FIG.3A shows the results of treatment with the calcium salt of the2,5dihydroxybenzenesulfonic acid. FIG. 3B show the results of treatmentwith the potassium salt of the 2,5dihydroxybenzenesulfonic acid;

FIG. 4 shows the images of an area of an experiment of a control culture(FIGS. 4A and 4B) and of another culture treated with the compound(FIGS. 4C and 4D);

FIG. 5 shows histograms of the experiments to evaluate the effect of the2,5-dihydroxybenzenesulfonic acid in the potentiation of the differentcytostatic medicines: Cisplatin (5 μg/ml) (FIG. 5A); Vincristine (0.1μl/ml) (FIG. 5B); Paclitaxel (5 μg/ml) ((FIG. 5C); and 5-fluorouracil(100 μg/ml) (FIG. 5D);

FIG. 6 shows images of a representative control experiment (FIG. 6A) andanother experiment in which the cells were treated during 24 hours withthe compound (FIG. 6B);

FIG. 7 is a graph representing the percentage data of all theexperiments showing that the 2,5-dihydroxybenzenesulfonic acid inhibitsup to 64% of migration of tumor cells;

FIGS. 8A and 8B show the images corresponding to an embryo treated with3 μg of bFGF+0, 1% heparin (FIG. 8A) and another embryo to which 100 μMof a potassium salt solution of the 2,5-dihydroxybenzenesulfonic acidwas added on the next day (FIG. 8B); and

FIG. 9 shows images before treatment, at six and at thirteen days aftertreatment of the same chronic psoriatic plaque located in the extensionarea of the left elbow treated with the potassium salt of the2,5-dihydroxybenzenesulfonic acid at 5%.

DETAILED DESCRIPTION

The 2,5-dihydroxybenzenesulfonic acid formulated in the form of salt isa commercial product (for example, the potassium salt may be acquired atMerck Farma y Quimica SA, Mollet del Vallés, Barcelona) with thefollowing molecular formula:

in which Met=Metal and n is a function of the metal valence used in thesalt. Generally n 0 1 or 2 for being the metal cation former of thesalt, univalent (K) or divalent (Ca óMg). The new biological activitiesof the 2,5-dihydroxybenzenesulfonic acid do not depend of the cationbond to the benzene ring because the 2,5-dihydroxybenzenesulfonic acidformulated with any salt has similar effects in the inhibition of cellproliferation, migration and angiogenesis. This invention only describesthe activities of the 2,5-dihydroxybenzenesulfonic acid formulated aspotassium and calcium salt without forgetting that within the scope ofthis invention is any pharmaceutically acceptable salt of the compound.The term “pharmaceutically acceptable salts” include metal salts oraddition salts which can be used in pharmaceutical forms. Thepharmaceutically acceptable salts of the 2,5-dihydroxybenzenesulfonicacid can be obtained from organic or inorganic acids or bases, throughconventional methods, by making the appropriate acid or base react withthe compound.

The pharmaceutical compositions containing the2,5-dihydroxybenzenesulfonic acid can be presented in any adequateadministration form, for example, systemic, oral, parenteral, urethral,rectal or topical administration, for which the necessarypharmaceutically acceptable excipients will be included for formulationof the desired form of administration.

The following examples illustrate and support the invention and shouldnot be considered as limitations of the scope of the invention.

Example 1 Illustrative Assay of the Anti-Proliferative Ability of the2,5-Dihydroxybenzenesulfonic Acid

This in vitro study, was carried out in three different triplicateexperiments with rat gliomic cells (C6 line). The cells were cultured ina medium formed by DMEM Dulbecco's modified Eagle's Medium (Gibco.Paisley UK), 7.5% of fetal serum (Gibco) 10 units/ml of penicillin(Gibco) and IO ug/ml of streptomycin (Gibco). The cultures were kept ina humid atmosphere at 3 TC. To evaluate the effect of the2,5-dihydroxybenzenesulfonic acid on the cell proliferation, 2×104 C6cells per ˜ell were seeded in 24-well (15 mm of diameter) plates. Theexperimental cultures were treated during 48 hours with different micromolar concentrations (μM) of the compound (calcium or potassium salt ofthe 2,5-dihydroxybenzenesulfonic acid). The controlled cultures lived 48hours, without adding the compound. Photographs of the cultures weretaken after 48 hours using an inverted microscope and then, the cultureswere colored with crystal violet (Merck Farma y Quimica SA. Mollet delVallés, Barcelona) and processed to determine the number of cells perwell, using a spectrum photometric method. As shown in FIG. 1, treatmentwith different concentrations of the compound produces a dose-dependentinhibition of cell proliferation, obtaining 88% inhibition with aconcentration of 100 μM of the calcium salt of the2,5-dihydroxybenzenesulfonic acid (A). With the same concentration ofthe potassium salt of the 2,5-dihydroxybenzenesulfonic acid, a 74%inhibition was obtained (B). The IC₅₀ is near to 25 μM for the calciumsalt and between 40 and 50 μM for the potassium salt. Comparing FIG. 1Awith FIG. 1B, it is observed that to obtain the same percentage ofinhibition in cell proliferation after treatment with the calcium saltof the compound, a double concentration of potassium salt is necessaryto obtain the same effect. This is due to the fact that the calcium saltof the compound contains two active principle moles(2,5-dihydroxybenzenesulfonic acid) that separate from salt in aqueoussolution. FIG. 2 shows the image of the C6 cells culture after 48 hourswithout treatment (A), another image corresponding to the C6 cellsculture treated for 48 hours with a concentration of 50 μM of thecalcium salt of the 2,5-dihydroxybenzenesulfonic acid (B) and a thirdone corresponding to a culture of C6 cells treated during 48 hours with100 μM of the potassium salt of the acid (C). This study shows that thetreatment with the compound inhibits proliferation in neoplastic cellsand corroborates the antiproliferative effect of the compound observedin normal vascular smooth muscular cells stimulated in vitro withmitogenic factors (Pares-Herbute N et al. Int J Angiol 8: 85-810, 1999).To distinguish if the anti proliferative activity of the2,5-dihydroxybenzenesulfonic acid is mediated by a cytotoxic or aproapoptotic effect, we conducted different experiments detailed in thefollowing example:

Example 2 Illustrative Assay of the Proapoptotic Ability of the 2,5Dihydroxybenzenesulfonic Acid

This assay was carried out with the C6 cells according to the proceduredescribed in example 1. To demonstrate the proapoptotic effect of theanalyzed compounds we have used two different methods that detect theintracellular fragmentation of the DNA and the apoptotic nuclei in situ.

Detection of the Intracellular Fragmentation of the DNA.

The enzymatic immunoassay methods to quantify the DNA fragmentsassociated to histones may be considered suitable to determine the onsetof apoptosis (Aragane Y et al. J Cell Biol 1998; 140: 171-182). Thismethod allows to differentiate death due to necrosis from death due toapoptosis since in necrosis the cytoplasmic membrane is fragmented andthe DNA appears in the culture medium, while in apoptosis, thefragmented DNA remains in the interior of the cell because the plasmamembrane remains intact (Aragane Y et al. J Cell Biol 140: 171-182,1998).

Using the Cell Death Detection ELISAP^(plus) kit (Boehringer Mannheim,Mannheim, Germany) in accordance with the manufacturer's instructions,we have determined the fragmentation of DNA in C6 (2×10³) cell culturesat 4, 16, 24 and 48 hours. The controlled cultures did not receive anytreatment while from 50 to 200 μM (FIG. 3A) of the potassium salt of the2,5-dihydroxybenzenesulfonic acid were added to the experimentalcultures. Experiments were also conducted adding from 25 to 100 μM ofthe calcium salt of the 2,5-dihydroxybenzenesulfonic acid (FIG. 3B). Allexperiments were performed in triplicate in three different experiments.

FIGS. 3A and 3B show the following: a) the antiproliferative effect ofthe 2,5-dihydroxybenzenesulfonic acid is mainly mediated by aproapoptotic activity; b) the cation bonded to the molecule does notdetermine the activity of the compound because the proapoptotic effectis similar using the calcium or potassium salt of the compound; c) thehighest proapoptotic effect is obtained in cells treated with thecompound during 48 hours; d) the maximum effect is obtained with aconcentration of 25 μM for the calcium salt and 50 μM for the potassiumsalt, identical to the IC₅₀ in cellular proliferation studies. Once itis proven that the antiproliferative mechanism of the2,5-dihydroxybenzenesulfonic acid participates in the cell death due toapoptosis, we quantitatively evaluated such effect through a microscopicstudy of gliomic cells using the following technique:

In Situ Detection of Apoptotic Nuclei (TUNEL Technique)

Three independent experiments were made, repeated three times. The C6cells from controlled cultures and those from cultures treated during 24hours with the (50 μM and 100 μM of the calcium and potassium saltsrespectively) were adhered to glass slides and fixed with a 4%paraformaldehyde buffered solution (pH 7.4) for one hour at thelaboratory temperature. Afterwards, the cells were washed andpermeabilized with a 0.1% solution of Triton X-100. Then the cells werewashed before applying the TUNEL technique [(terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick and labeling (Gavrieli Y, ShermanY, Bensasson S A. J Cell Biol 119: 493-501, 1992). A kit for in situdetection of apoptotic nuclei (In situ Cell Death Detection KitBoehringer Mannheim, Mannheim, Germany) was used. The different stagesof the technique were followed in accordance with the instructions ofthe kit manufacturer. Finally, the cells were colored with green light(Fluka, AG, Switzerland). The TUNEL reaction only appears in theapoptotic nuclei.

Although very similar results were obtained with the calcium andpotassium salt of the compound, object of the invention, only theresults obtained with the potassium salt of the compound are shown.Cells were counted in 6 different fields in twelve slides where thecells from the 6 control cultures and the 6 cultures treated with the2,5-dihydroxybenzenesulfonic acid (100 μM) had adhered. The total numberof non apoptotic and apoptotic cells was as follows:

C6 Cells Apoptotic Nuclei Normal Nuclei Control Cells 138 5954 TreatedCells 3846 354

The total number of treated cells is lower than the total number ofcontrol cells due to the antiproliferative effect of the compound.

The images of FIG. 4 show an area of an experiment of a control culture(A and B) and of another culture treated with the compound (C and D) inwhich the TUNEL technique was employed. As shown in the images, only twoapoptotic nuclei are observed on the control cells while in the treatedcells with the compound object of the invention there are 107 apoptoticnuclei and only 8 normal nuclei (non apoptotic).

These data show that the 2,5-dihydroxybenzenesulfonic acid is a compoundwith an important proapoptotic activity useful to induce tumorapoptosis. Given that it has been proven that the2,5-dihydroxybenzenesulfonic acid inhibits apoptosis in normal humancells (Braber R, Farine J C, Lora G A. Apoptosis 4: 4111-49, 1998), thiscompound is a strong molecule candidate for treatment of cancer.

One of the mechanisms involved in the therapeutic failure ofchemotherapy and radiotherapy is the inefficacy of these treatments toinduce cellular death by apoptosis, mainly due to the hyper expressionof antiapoptotic proteins in tumor cells (Sellers W R, Fisher Del. JClin Invest 104: 1655-1661, 1999; Branch P. et al. Oncogene 19:3138-3145, 2000). Therefore, the proapoptotic compounds may be of greatclinical use as an adjuvant in chemotherapy and radiotherapy treatments.

Once the proapoptotic effect of the 2,5-dihydroxybenzenesulfonic acidwas demonstrated, we evaluated the ability of this compound to increasethe antiproliferative effect of the different cytostatic medicines. Thefollowing example demonstrates how the 2,5-dihydroxybenzenesulfonic acidis capable of increasing the therapeutic efficacy of the differentcytostatic compounds used in oncology such as cisplatin, vincristine,paclitaxel and 5-fluorouracil.

Example 3

Illustrative Assay of the Ability of the 2,5-Dihydroxybenzene SulfonicAcid in Potentiation of Chemotherapy

We used for this study C6 cells cultured in vitro under the sameconditions described in example 1. 1×103 cells per well were cultured in24-well plates. Three types of treatment were made: a) 24 hours afterthe seeding, the cells were separately treated with each one of thefollowing medicines; cisplatin (5 μg/ml), vincristine (0.1 μg/ml),paclitaxel (5 μg/ml) and 5-fluorouracil (100 μg/ml); b) 24 hours afterthe seeding, the cells were treated jointly with the2,5-dihydroxybenzenesulfonic acid (potassium salt, 100 μM) and with eachone of the following medicines; cisplatin (5 μg/ml) vincristine (0, 1μg/ml), paclitaxel (5 μg/ml) and 5-fluorouracil (100 μg/ml); c) at thetime of the seeding (Day 0), the cells were pre-treated with the2,5-dihydroxybenzenesulfonic acid (potassium salt, 100 μM). Next day thecultures were treated also with each one of the following medicines:cisplatin (5 μg/ml) vincristine (0, 1 μg/ml), paclitaxel (5 μg/ml) and5-fluorouracil (100 μg/ml). The controlled cultures did not receivetreatment for 2 days. After 48 hours (day 2), the cells of identicalshape to the ones used in example 1 were evaluated in all the cultures.This study was carried out in triplicate independent experimentsrepeated three times.

FIGS. 5 (A, B, C, and D) shows the histograms of the experimentsperformed to evaluate the effect of the 2,5-dihydroxybenzenesulfonicacid in the potentiation of the different 5 cytostatic medicines.Treatment with cisplatin, vincristine and 5-fluorouracil produces aninhibition of 50% in proliferation of C6 cells, while the treatment withpaclitaxel obtains 67% of inhibition of the cellular proliferation. Thecombined treatment of the 2,5-dihydroxybenzenesulfonic acid+thecytostatic medicines (cysplatin, vincristine and 5-fluorouracil)produces an inhibition of 84% in cellular proliferation. The combinedtreatment with 2,5-dihydroxybenzenesulfonic acid+paclitaxel produces 86%in the inhibition of the cellular proliferation. When cellular culturesare pre-treated with the 2,5-dihydroxybenzenesulfonic acid andafterwards with the following cytostatic medicines: cisplatin,vincristine and 5-fluorouracil, an inhibition of 90% is obtained in thecell proliferation. When paclitaxel is used, the inhibition in cellularproliferation reaches up to 92%.

The above mentioned results demonstrate that the simultaneous treatmentof the 2,5-dihydroxybenzenesulfonic acid with the chemical therapyagents, increases their therapeutic efficacy and besides this chemicalpotentiation effect is higher when the cells has been pre-treated withthe 2,5-dihydroxybenzenesulfonic acid. These data support the use of the2,5-dihydroxybenzenesulfonic acid as an adjuvant in the treatmentassociated with chemical therapy and radiotherapy.

Example 4

Illustrative Assay of the Antimigration Ability of the 2,5Dihydroxybenzenesulfonic Acid

This assay was carried out in three different triplicate experiments. Toevaluate the ability of the 2,5-dihydroxybenzenesulfonic acid in theinhibition of cellular migration C6 2×10⁵ cells cultured in vitro in 20mm plates were used. A longitudinal lesion was made with a sterilemicropipette (day 0) to the control cultures and in cultures treatedwith 100 μM of the potassium salt of the 2,5-dihydroxybenzenesulfonicacid. Digital photos were taken using a photographic system connected toa luminous microscope and the area of the lesion was delimited using acomputerized morphometric program (Moticam. Motic. Barcelona).

Photographs were taken again after 24 hours, and the borders of thelesion were marked overlapping the first two photos (day 0) with thoseobtained after 24 hours to calculate the percentage of the injured areacovered by the migratory cells. These values were represented as apercentage of the regeneration obtained with the treatment. FIG. 6 showsa typical example of a control experiment (A) and another experiment inwhich the cells were treated during 24 hours with the compound object ofthe invention (B). As observed in this Figure, the non treated cellscompletely regenerate the lesion (FIG. 6A) while the cells treated withthe compound are not capable of migrating and cover all the area of thelesion (FIG. 6B). FIG. 7 that represents the percentage data of all theexperiments shows that the 2,5-dihydroxybenzenesulfonic acid inhibits upto 64% of migration of tumor cells.

Example 5

Illustrative Assay of the Antiangiogenic Ability of the2,5-Dihydroxybenzenesulfonic Acid

We used for this assay the chorioallantoic membrane of a chick embryofor testing the activity of antiangiogenic substances in vivo(Zilberberg L. et al. J Biol Chem 2003; 278: 35564-35573). We used aproangiogenic compound, the basic form of the fibroblast growth factor(bFGF) (Meghna U et al. Blood 2003; 102: 2108-2114).

Fertilized eggs are kept in a incubator at 37° C. with a humidity of80%. After 4 days, a hole is made in the narrowest end of the egg shellto collect 1 ml of albumin Then, the hole is covered with a paraffinfilm (Parafilm M Laboratory Film Chicago Ill. USA). This procedureallows creating an air chamber that prevents the embryo to adhere to theupper part of the shell. On day 13 of incubation, the shell is split atthe air chamber level to perform the treatment. Twenty embryos aretreated with 5 μl of a solution of 3 μg of bFGF+0.1% heparin, soaked ina nitrocellulose paper disc. Afterwards the shell is sealed with aparaffin film. Next day, in half of the embryos (n=10) the shell isuncovered to soak again the nitrocellulose paper disc with 100 μM ofpotassium salt of the 2,5-dihydroxybenzenesulfonic acid dissolved inphysiological saline (5 μl). The hole in the shell is then covered againwith a paraffin film. On day seventeen the experiment ends, takingphotographs of the nitrocellulose piece for the comparison study.

FIG. 8 presents two images corresponding to an embryo treated with 3 ugof bFGF+0, 1% heparin (A) and another embryo to which 100 J.!M of apotassium salt solution of the 2,5-dihydroxybenzenesulfonic acid wasadded on the next day (B) Image A shows how the nitrocellulose disc isinvaded by blood vessels while Image B shows a very scarce vascularinvasion in the disc. The morphometric quantification of the images ofthe nitrocellulose discs using a computerized system (Moticam Motic.Barcelona) shows the antiangiogenic effect of the compound (area of thedisc covered by blood vessels in embryos treated withbFGF+heparin=35±8.6% vs. area of the disc covered by blood vessels inembryos treated with bFGF+heparin+potassium salt of the2,5-dihydroxybenzene sulfonic acid=2±1.5%; p<O,OOOI; unpaired student'st-test). Similar effects were obtained using 50 μM of the calcium saltof the compound.

This experiment shows that the compound object of this invention has anantiangiogenic activity for being capable of neutralizing the angiogeniceffect induced by bFGF.

Example 6 Assay on Psoriatic Lesions

We used for this study the potassium salt of the2,5-dihydroxybenzenesulfonic acid formulated at 2.5 and 5% in cream forbeing this type of formulation a usual procedure for topical treatmentof skin diseases. The selected concentrations of the salts of the2,5dihydroxybenzenesulfonic acid are within the range of theconcentrations used for treatment of diabetic retinopathies: 6 tabletsper day of 500 mg of calcium salt of the 2,5-dihydroxybenzenesulfonicacid (Benakis A et al Therapie 1974; 29: 211-219). As aqueous phase ofthe cream we have used distilled water. The fatty phase can beconstituted by cetylic alcohol, stearic alcohol or vaseline. The span isan efficient emulsifier in the preparation of the cream. Although bothformulations (2.5 and 5%) of the product show to be clinicallyefficient, the best therapeutic benefit is obtained with theconcentration at 5%. The following example illustrates the formulationof an efficient cream for the topic treatment of psoriasis, by way ofexample and not of limitation of the scope of the invention.

I.—Active Part (potassium salt of the 2,5-dihydroxybenzenesulfonic acidat 5.6%)II.—Inactive Part (as excipients cetylic alcohol (2.5%), stearyl alcohol(2.5%), liquid vaseline (30%), white soft paraffin (20%), sorbitanoleate (5%) and distilled water (c.s.p 0.100 g).

The clinical efficacy of the treatment was evaluated according to theindex that quantifies the desquamation signs (D), erythema (E) andinfiltration (I) to which the following assessment was assigned: (0)absent; (1) slight; (2) moderate and (3) severe (Freeman A K et al. J.Am. Acad Dermat 2003; 48: 564-568). FIG. 9 shows three images: beforetreatment, six and thirteen days after treatment of the same chronicpsoriatic plaque located in the extension area of the left elbow treatedwith the potassium salt of the 2,5-dihydroxybenzenesulfonic acid at 5%.As can be observed, the topical treatment two times at day with a creamcontaining the potassium salt of the 2,5-dihydroxybenzene sulfonic acidproduces an early (6 days) very notable “clearance” of the plaque withalmost total disappearance of hyperkeratosis. The therapeutic efficacyof the cream is more evident at the end of the second week of treatment.The treatment produces a significant reduction of the global values ofthe DEI index (DEI global pre-treatment=6±1.57 vs. DEI globalpost-treatment=1±0.58; p<0.0001; unpaired student's r-test).

Figures Captions

1. Histogram showing the anti proliferative effect of the treatment withdifferent concentrations of the (A) calcium and (B) potassium salts ofthe 2,5-dihydroxybenzenesulfonic acid in cultures of C6 cells after 48hours of treatment. Ordinates: Absorbance at 595 nm; Abscises:concentration: μM.

2. Panel A shows the aspect of a control culture of C6 cells after 48hours. Panel B shows an image of a culture of C6 cells treated during 48hours with 50 μM of the 2,5-dihydroxybenzenesulfonic acid (calciumsalt). Panel C shows a culture of C6 treated during 48 hours with 100 μMof the potassium salt of the 2,5dihydroxybenzenesulfonic acid.

3. Representative histograms in which it is observed that theantiproliferative effect of the 2,5-dihydroxybenzenesulfonic acid is notdue to necrosis (white histogram) but to apoptosis (lined histogram). A:treatment with the calcium salt of the 2,5-dihydroxybenzenesulfonicacid. B: Treatment with the potassium salt of the2,5-dihydroxybenzenesulfonic acid. Ordinates: Absorbance at 405 nm;Abscises: time in hours.

4. Images of gliomic C6 cells processed with the TUNEL technique forin-situ detection of apoptotic cells. The apoptotic nuclei are showndark and the nucleus and cytoplasm of the cell of the non apoptoticcells are shown in light color. The arrows indicate apoptotic nucleus. Aand B control cells, C and D cells treated with2,5-dihydroxybenzenesulfonic acid. Photographs Band D correspond to azoom of the boxes of A and C photographs respectively.

5. Histograms demonstrating the potentiating effect on chemotherapy(assessed as an anti proliferative effect) of the2,5-dihydroxybenzenesulfonic acid, with different cytostatic compoundsA) Cisplatin (5 ug/ml); B) Vincristine (0.1 μl/ml); C) Paclitaxel (5Ug/ml) and D) 5-fluorouracil (100 j-tglml). Ordinates: Absorbance 595nm; Abscises: white histogram (control); dotted (cytostatic; day 1);lined histogram (2,5-dihydroxybenzenesulfonic acid+cytostatic; day 1);square histogram (2,5-dihydroxybenzenesulfonic acid (day 0)+cytostatic;day 1).

6. Photographic images of cellular migration in a control experiment andother experiments where the cells were treated with the2,5-dihydroxybenzenesulfonic acid (B). The control cells totallyregenerate one lesion made during the culture, while the cellularmigration of the cells treated with the 2,5dihydroxybenzenesulfonicacid, was unable to fully cover the affected area of the culture. Thehorizontal lines delimit the initial longitudinal lesion made in thecultures.

7. Histogram representing the migratory ability of the C6 cells incontrolled cultures (white histogram) and in cultures treated with the2,5-dihydroxybenzenesulfonic acid (black histogram). The migratoryability is expressed (ordinates) as a percentage of regeneration(percentage of the area covered of a longitudinal lesion made in thecultures)

8. Images of two chicken embryos with 17 days of incubation. Panel Acorresponds to an embryo treated with 3 μg of bFGF+0, 1% of heparin.Panel B shows the aspect of an embryo treated with 3 μg of bFGF+0.1% deheparin+100 μM of the potassium salt of the 2,5-dihydroxybenzenesulfonicacid. Panel A shows the antiangiogenic effect of the2,5-dihydroxybenzenesulfonic acid because the nitrocellulose disc usedas releasing vehicle of the substance appears almost without anyvessels.

9. Images of a hiperkeratosic psoriatic plaque located in the rearregion of the left elbow. Image A represents the aspect of the psoriaticplaque before initiating treatment. Image B is an aspect of the sameplaque after six days of treatment with a cream at 5% containing as anactive component the potassium salt of the 2,5-dihydroxybenzenesulfonicacid. Image C shows the aspect of the psoriatic plaque after two weeksof treatment with the potassium salt of the 2,5dihydroxybenzenesulfonicacid formulated at 5%. The numbers shown in the images correspond to theday on which the photographs were taken.

What is claimed is:
 1. A method of treating an angiodependent disease, comprising administering 2,5-dihydroxybenzenesulfonic acid, or a salt thereof with a pharmaceutically acceptable inorganic or organic acid or base.
 2. The method of claim 1, wherein a salt of 2,5-dihydroxybenzenesulfonic acid with an inorganic or organic base is administered.
 3. The method of claim 1, wherein a salt of 2,5-dihydroxybenzenesulfonic acid with an inorganic base is administered.
 4. The method of claim 1, wherein a potassium, calcium, or magnesium salt of 2,5-dihydroxybenzenesulfonic acid is administered.
 5. The method of claim 1, further comprising administering a cytostatic medicine.
 6. The method of claim 1, wherein an angiodependent disease is psoriasis.
 7. The method of claim 1, wherein 2,5-dihydroxybenzenesulfonic acid or a salt is administered systemically.
 8. The method of claim 1, wherein 2,5-dihydroxybenzenesulfonic acid or a salt is administered topically.
 9. A method of reducing psoriasis hyperkeratosis, comprising administering 2,5-dihydroxybenzenesulfonic acid, or a salt thereof with a pharmaceutically acceptable inorganic or organic acid or base.
 10. The method of claim 9, wherein 2,5-dihydroxybenzenesulfonic acid or a salt is administered topically.
 11. The method of claim 9, wherein a salt of 2,5-dihydroxybenzenesulfonic acid with an inorganic or organic base is administered.
 12. The method of claim 9, wherein a salt of 2,5-dihydroxybenzenesulfonic acid with an inorganic base is administered.
 13. The method of claim 9, wherein a salt of 2,5-dihydroxybenzenesulfonic acid with an inorganic base is administered topically.
 14. The method of claim 9, wherein a potassium, calcium, or magnesium salt of 2,5-dihydroxybenzenesulfonic acid is administered.
 15. A method of reducing psoriasis hyperkeratosis, comprising topically administering a potassium, calcium, or magnesium salt of 2,5-dihydroxybenzenesulfonic acid. 